E1A ACTIVATION OF INSULIN-RECEPTOR GENE-EXPRESSION IS MEDIATED BY SP1-BINDING SITES

E1a adenoviral oncoproteins have been known to modulate genes important for the growth and differentiation of cells. Our laboratory is interested in understanding how insulin promotes the growth and proliferation of cells. In this report, we have examined the ability of E1a to modulate the insulin receptor gene expression. In HepG2 cells, expression of the 243-amino acid E1a protein stimulated expression of the chloramphenicol acetyltransferase reporter under the control of the insulin receptor promoter. 5'-Deletion analysis of the insulin receptor promoter indicated that the region between -630 and -607 is important for regulation by E1a. This region contains two GA and four overlapping GC boxes that are putative Sp1-binding sites. A DNA fragment containing these sites was used as a probe in gel retardation assays. Three specific protein-DNA complexes were detected with HepGP nuclear extract. These complexes could be competed partially by the DNA fragments with mutations in either the GA or GC boxes, but not by the DNA fragment with a mutation in both the GA and GC boxes. In addition, mutation of each of these sites lowered the basal activity of the promoter and partially reduced transactivation by E1a. Simultaneous mutation in both GA and GC boxes further reduced the basal activity and abrogated transactivation by E1a. Taken together, these results indicate that the loss of binding ability of Sp1 (or Sp1-like factors) is concomitant with reduction of the basal activity and the loss of E1a inducibility of the gene. Therefore, the GA and GC boxes are responsible for transactivation of the insulin receptor gene promoter by E1a. We also propose that transactivation of insulin receptor gene by E1a is mediated by Sp1 transcription factor.